Anat Cell Biol.  2011 Jun;44(2):143-150. 10.5115/acb.2011.44.2.143.

Immunohistochemical localization of cyclic AMP-responsive element binding protein (CREB)-binding protein in the pig retina during postnatal development

• Oh H
• Kim H
• Ahn M
• Jeong C
• Jeong J
• Moon C
• Shin T

• KMID: 1447424
• DOI: http://doi.org/10.5115/acb.2011.44.2.143

Abstract

This study evaluated the cellular localization of cyclic AMP-responsive element binding protein-binding protein (CBP) expression in pig retinas during postnatal development. Immunohistochemistry and Western blot analysis were performed on retinal tissue from 2-day-old, 5-week-old, and 6-month-old pigs. Western blot analysis detected the expression of CBP in the retinas of 2-day-old piglets and showed that it was significantly decreased in the retinas of 5-week-old and 6-month-old pigs. Immunohistochemically, CBP was intensely immunostained in protein kinase C alpha (PKCalpha)-positive-bipolar cells, glutamine synthetase-positive Muller cells, and in ganglion cells in 2-day-old piglets. CBP was detected weakly in the inner plexiform, outer nuclear, and rod and cone layers. CBP immunoreactivity in the ganglion cell layer was decreased in the retinas of 5-week-old and 6-month-old pigs, while clear CBP expression detected in the neurite of PKCalpha-positive bipolar cells in the inner nuclear layer. In addition, CBP immunoreactivity in Muller cells and glial fibrillary acidic protein-positive glial processes was particularly noteworthy in pig retinas, but not in rat retinas. The results indicate that CBP is expressed differentially in the retinal neurons and glial cells according to growth and animal species, and may play an important role in homeostasis in Muller cells, neurite extention in bipolar cells, and signal transduction in photoreceptor cells in the porcine retina.

Keyword

Cyclic AMP-responsive element binding protein-binding protein; Retina; Pig; Muller cells; Development

MeSH Terms

Animals
Blotting, Western
Carrier Proteins
Ganglion Cysts
Glutamine
Homeostasis
Humans
Immunohistochemistry
Infant
Neurites
Neuroglia
Photoreceptor Cells
Protein Kinase C-alpha
Rats
Retina
Retinal Neurons
Retinaldehyde
Signal Transduction
Swine
Carrier Proteins
Glutamine
Protein Kinase C-alpha
Retinaldehyde

Figure

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  Fig. 1 Western blot analysis of cyclic AMP-responsive element binding protein-binding protein (CBP) in the retinas of 2-day-old, 5-week-old, and 6-month-old pigs. (A) A representative photograph of a Western blot for CBP and β-tubulin. CBP and β-tubulin are detected at adove 200 kDa and at approximately 55 kDa, respectively, as confirmed by molecular weight markers. (B) Bar graph of densitometric data analysis (mean±standard error, n=4-5 pigs/group). The relative levels of CBP are calculated after normalization to β-tubulin bands. The value indicating CBP expression levels from the retina at 2-day-old is arbitrarily defined as 100 (B, bar graphs). *P<0.05 vs. 2-day-old piglets.

• 

  Fig. 2 Immunohistochemical analysis of cyclic AMP-responsive element binding protein-binding protein (CBP) in the retinas of 2-day-old (A, E, H), 5-week-old (B, F, L) and 6-month-old pigs (C, G, J). CBP immunoreactivity is detected in glial processes (arrowheads) and ganglion cells (arrow) in the ONF and GCL, some glial processes in the IPL, neuronal cells in INL, OPL, and RCL in the retinas of 2-day-old (A, E, H), 5-week-old (B, F, L) and 6-month-old pigs (C, G, J). (E and H, F and I, and G and J) are high-magnification images of GCL, and INL in (A, B, and C), respectively. No specific reaction product is seen in sections incubated with non-immune sera (D). In the rat retina, CBP immunostaining is evident in the ganglion cells (arrow) in the GCL, and some cells in the INL (K). Note the localization of CBP immunoreactivity mainly in the cytoplasm, the nucleus in the pig, and the retina in the rat. The retinal layers are indicated to the right: ONF, optic nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RCL, layer of rods and cones. Counterstained with hematoxylin. Scale bars=25 µm (A-D, K), 12 µm (E-J).

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  Fig. 3 Immunofluorescence staining of cyclic AMP-responsive element binding protein-binding protein (CBP, TRITC) with glial fibrillary acidic protein (GFAP, FITC) in the retina of 2-day-old, 5-week-old, and 6-month-old pigs. GFAP is detected very rarely in the retina of 2-day-old piglets, and it is abundant in the retina of 5-week-old and 6-month-old pigs. CBP immunoreactivity is occasionally co-localized in the GFAP-positive glial cells. ONF, optic nerve fiber layer; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RCL, layer of rods and cones. Scale bars=50 µm.

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  Fig. 4 Immunofluorescence staining of cyclic AMP-responsive element binding protein-binding protein (CBP, TRITC) with glutamine synthetase (GS, FITC) in the retina of 2-day-old, 5-week-old, and 6-month-old pigs. In ganglion cell layer (GCL), GS immunoreactivity is more abundant in retina of 2-day-old piglets compared to that of 5-week-old and 6-month-old pigs. CBP immunofluorescence is co-localized in GS-positive Müller cells. ONF, optic nerve fiber layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RCL, layer of rods and cones. Scale bars=50 µm.

• 

  Fig. 5 Immunofluorescence staining of cyclic AMP-responsive element binding protein binding protein (CBP, TRITC) with protein kinase C, alpha (PKCα, FITC) in the inner nuclear layer (INL) of retina of 2-day-old, 5-week-old, and 6-month-old pigs. CBP immunofluorescence is co-localized in PKCα-positive bipolar cells. INL, inner nuclear layer. Scale bars=20 µm.

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